Use of non-agrobacterium bacterial species for plant transformation

ABSTRACT

The invention relates to methods for Rhizobia-mediated genetic transformation of plant cells, including soybean, canola, corn, and cotton cells. These include both VirD2-dependent and VirD2-independent methods. Bacterial species utilized include strains of  Rhizobium  sp.,  Sinorhizobium  sp., and  Mesorhizobium  sp. Vectors for use in such transformation are also disclosed.

This application claims the priority of U.S. Provisional PatentApplication 60/800,872, filed May 16, 2006, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of plant biotechnology. Inparticular, the invention relates to methods for producing transgenicplants and plant cells by using non-Agrobacterium bacterial species.

2. Description of Related Art

Agrobacterium spp., members of the Rhizobiales, are common soilbacteria, along with Rhizobium spp., Mesorhizobium spp., Sinorhizobiumspp., and related species and genera. A number of wild-type and disarmed(non-pathogenic) strains of Agrobacterium tumefaciens and Agrobacteriumrhizogenes harboring Ti or Ri plasmids can be used for gene transferinto plants. Phytohormone synthesis genes located in the T-DNA of wildtype Agrobacteria harboring a Ti or Ri plasmid are expressed in plantcells following transformation, and cause tumor formation or a hairyroot phenotype depending on the Agrobacterium strain or species.Importantly, T-DNA of Agrobacteria can be engineered to replace many ofits virulence and pathogenicity determinants with “genes of interest”while retaining the ability to be transferred into a plant cell andintegrated into a plant genome. Strains containing such “disarmed” Tiplasmids are widely used for plant transformation.

The mechanism of T-DNA transfer to plant cells by Agrobacterium has beenwell documented. Briefly, the T-DNA is delimited by two border regions,referred to as right border (RB) and left border (LB). The borders arenicked by virulence protein VirD2 which produces single strandedtransferred DNA (the “T-strand”) with covalent attachment of the VirD2on its 5′ end. The protein-DNA complex, also including AgrobacteriumVirE2 protein, exits Agrobacterium cells through the so-called Type 4secretion system (T4SS, both virulence protein and ssDNA transporter),and is transferred into plant cells and integrated in the plant genomewith the help of both Agrobacterium virulence proteins and plantfactors. The use of Agrobacterium-mediated vectors to introduce DNA intoplant cells is well known in the art. See, for example, the methodsdescribed by Fraley et al., (1985), Rogers et al., (1987) and U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety.

Agrobacterium-mediated transformation is efficient in manydicotyledonous plants including Arabidopsis, tobacco, and tomato.Methods for Agrobacterium-mediated transformation of other species havealso been devised (e.g. U.S. Pat. No. 6,384,301, relating to soybeantransformation). While Agrobacterium-mediated transformation was atfirst only used with dicotyledonous plants, advances inAgrobacterium-mediated transformation techniques made the techniqueapplicable to monocotyledonous plants as well. For example,Agrobacterium-mediated transformation techniques have been applied torice (Hiei et al., 1997; Zhang et al., 1997; U.S. Pat. No. 5,591,616,specifically incorporated herein by reference in its entirety), wheat(McCormac et al., 1998), barley (Tingay et al., 1997; McCormac et al.,1998), and maize (Ishida et al., 1996). However, a number of plantspecies are recalcitrant to Agrobacterium-mediated transformation, andefficiency is low in others. Additionally, since A. tumefaciens entersplant tissues at wound sites, and does not naturally infect unwoundedtissues, the use of certain tissues as transformation targets is notavailable.

Besides the T4SS-dependent T-strand delivery system, Agrobacterium hasadditional plasmid mobilization systems that can also transfer andintegrate plasmids, such as the IncQ plasmid pRSF1010, between bacterialcells and into the plant genome with lower frequency via conjugaltransfer (Buchanan-Wollaston et al. 1987, Shadenkov et al. 1996; Chen etal., 2002). For example, the conjugal transfer protein MobA, inconjunction with MobB and MobC proteins of the RSF1010 plasmid, cleavesthe oriT (origin of transfer) site, attaches to the 5′ end and transfersthe ssDNA into cells independent of the T4SS system (Bravo-Angel et al.1999 and references therein).

Conjugal transfer systems are widely present in bacteria, resulting inexchange of genetic information between bacterial cells. In Rhizobium,phylogenetically related but distinct from Agrobacterium (Spaink, etal., (ed.), 1998; Farrand et al., 2003), the conjugal transfer systemhas been partially characterized in some species (Freiberg et al., 1997;Turner et al. 2002, Tun-Garrido et al. 2003, Perez-Mendoza et al. 2004).The conjugal system requires an oriT as the nicking site and TraA or Mobas a nicking enzyme, which is different from the conventional elementsused in T-DNA mobilization (VirD2 and RB and LB sites, respectively).Unlike VirD2, which was found to have plant NLS (nuclear localizationsignal) at its C-terminus for plant nuclear targeting, the TraA or Mobdoesn't have an obvious NLS. The precise mechanism and site ofintegration of DNA in plants by TraA remains unclear.

Members of the Rhizobiales other than Agrobacterium sp., such asRhizobium spp., are known to symbiotically associate with plant roots inspecialized nitrogen-fixing nodules (e.g. Long, 2001). In addition tohost-specific nodulation of plant roots, especially of legumes, someplant growth promoting effects by members of the Rhizobiales are knownin the absence of nodulation (e.g. Noel et al., 1996). Recently, reportshave been published describing transformation of plants by bacteriaother than Agrobacterium sp. (e.g. Broothaerts et al., 2005; U.S. PatentApplication Publications 20050289667; 20050289672; Weller et al., 2004;Weller et al, 2005).

Broothaerts et al., reported transformation by Rhizobium sp.,Mesorhizobium loti, and Sinorhizobium meliloti strains that was limitedto Arabidopsis, tobacco, and rice. Weller et al. (2004, 2005) reportedthat several bacteria, including strains of Rhizobium sp. andOchrobactrum sp. that harbored Ri plasmids apparently transformedhydroponically grown cucumber and tomato plants, leading to a hairy rootphenotype. However the presence of Agrobacteria was not ruled out as apossibility in some inoculated plants, complicating the analysis.Transfer of DNA to soybean, corn, cotton, or canola plant cells bynon-Agrobacterium bacterial strains has not been reported. In addition,transformation efforts in rice, tobacco, and Arabidopsis withnon-Agrobacterium bacterial strains have to date been hampered by lowtransformation efficiencies. There is, therefore, a great need in theart for the development of improved methods allowing the transformationof important crop species using non-Agrobacterium bacterial strains, andimproving transformation efficiencies in general.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1: Schematic map of pMON96033.

FIG. 2: Schematic map of pMON96036.

FIG. 3: Schematic map of pMON101316.

FIG. 4: Transient GUS assay of Rhizobium-mediated transformation insoybean with Mesorhizobium loti (ML), Rhizobium leguminosarum (RL),Sinorhizobium fredii (SF), Sinorhizobium meliloti (SM) with eitherdisarmed Ti-plasmid (pTiBo542G or pTi4404kan). RL4404: R. leguminosarumstrain Madison with pTi4404kan; ML542G: M. loti USDA3471 with pTiBo542G;ML4404: M. loti USDA3471 with pTi4404kan; 2370LBA: R. leguminosarumUSDA2370 with pTi4404kan; 2370G: R. leguminosarum USDA2370 withpTiBo542G; SF4404: S. fredii USDA205 with pTi4404kan; SM542C: S.meliloti USDA1002 with pTiBo542G; ABI: A. tumefaciens ABI straincontrol.

FIG. 5: Germline transmission of gus transgene in soy produced throughRhizobium-mediated transformation.

FIG. 6: Schematic map of pMON96913.

FIG. 7: Schematic map of pMON96914.

FIG. 8: Schematic map of pMON96026.

FIG. 9: Rhizobia-mediated transformation of canola with several strainsas shown by GUS transient assay. A) ML542C (22.4%); B) RL2370G (33.3%);C) RL2370LBA (20%); D) SF542C (30.5%); E) SF4404 (20.6%); and F) SM542C(13%). The % of explants with GUS positive sectors are shown inparentheses.

FIG. 10: Stable transgenic canola calli transformation with severalstrains of Rhizobia. A) ML542C (50%); B) RL2370G (21%); C) RL2370LBA(67%); D) SF542C (36%); and E) SM542C (73%). The % of explants with GUSpositive sectors are shown in parentheses.

FIG. 11: Southern blot detection of the CP4 transgene in canola plantsderived from Rhizobium-mediated transformation. Lane 1: BN_A22 line;lane 2: BN_A24 line; lane 3: BN_A28 line; and lane 4: BN_A35 line.

FIG. 12. Cotton transformation by Rhizobia containing pMON101316: A)ML542C (47.8%); B) RL2370G (56%); C) RL2370LBA (31.4%); D) SF542C(23.2%); E) SF4404 (31.5%); and F) SM542C (44.4%). RL2370 was used as anegative control; Agrobacterium tumefaciens ABI strain was used as apositive control. The percentage of GUS staining positive explants arewritten in parentheses above.

FIG. 13: Stable transformation of cotton calli by several Rhizobiastrains: A) ML542C; B) SF542C; C) SM542C; D) SF4404; E) RL2370LBA; andF) RL2370G.

FIG. 14: Detection of the gus transgene by Southern hybridization incotton calli derived from Rhizobium-mediated transformation. RL2370LBA:R. leguminosarum 2370 with LBA4404 Ti helper plasmid; SF542:Sinorhizobium fredii 205 with pTiBo542 helper plasmid from AGL0 strain;and SF4404: Sinorhizobium fredii 205 with LBA4404 Ti helper plasmid.

FIG. 15: Rhizobia-mediated corn transformation as shown by transientexpression of a gus gene in corn immature embryos. ABI: A. tumefaciens;RL2370LBA: Rhizobium leguminosarum USDA2370 with LBA4404 Ti plasmid;SM542C: Sinorhizobium meliloti USDA1002 with pTiBo542; ML542G:Mesorhizobium loti with pTiBo542; SF4404: Sinorhizobium fredii USDA205with LBA4404 Ti plasmid; SF542C: Sinorhizobium fredii USDA205 withpTiBo542. All strains contained pMON96036 and were induced in ATA mediumpH5.4.

FIGS. 16A-16C: Corn calli expressing the gfp marker after transformationwith Rhizobia strains.

FIG. 17: Southern hybridization analysis of transgene integration incorn plants derived from Rhizobium-mediated transformation. DIG-labeledgus probe was used to detect the transgene. Lane 1-2 and 11-12: linesderived after transformation with M. loti ML542G/pMON96036; lane 3-9:lines derived after transformation with A. tumefaciens ABI control; lane13-17: lines derived after transformation with S. frediiSF4404/pMON96033; Lane 18-19: lines derived after transformation with S.fredii SF542C/pMON96036.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for transforming a plantcell, comprising: (a) contacting at least a first plant cell with abacterium other than Agrobacterium sp. comprising: (i) a first nucleicacid comprising a vir gene region of a Ti plasmid wherein the vir generegion acts to introduce a nucleic acid coding for a sequence ofinterest into the plant cell in a VirD2-dependent manner; and (ii) asecond nucleic acid comprising one or more T-DNA border sequence(s)operably linked to a nucleic acid of interest; and (b) selecting atleast a first plant cell transformed with the nucleic acid of interest,wherein the plant cell is a soybean, canola, corn, or cotton plant cell.

In another aspect, the invention provides a method for transforming aplant cell, comprising: (a) contacting at least a first plant cell witha bacterium other than Agrobacterium comprising (i) a first nucleic acidrequired for conjugative transfer of DNA sequences independent of VirD2function, and (ii) a second nucleic acid comprising a nucleic acid ofinterest; wherein the plant cell is a soybean, canola, corn, or cottonplant cell and wherein polypeptides encoded by the nucleic acid requiredfor conjugative transfer act to transfer the nucleic acid of interestinto the plant cell; and (b) selecting at least a first plant celltransformed with the nucleic acid of interest. In such a method, theconjugative transfer may be traA, traI, or mobA-dependent, and the firstnucleic acid comprises oriT. The first nucleic acid may lack left andright T-DNA border sequences.

In a method of the invention, the bacterium may be Rhizobia cell. Incertain embodiments, the Rhizobia is grown under suitable conditions tominimize polysaccharide production by the Rhizobia cells. The Rhizobiacell may be grown in the presence of acetosyringone or other compound,such as a phenolic compound, that induces vir gene function prior tocontacting the plant cell. The Rhizobia cell may be selected from thegroup consisting of: Rhizobium spp., Sinorhizobium spp., Mesorhizobiumspp., Phyllobacterium spp. Ochrobactrum spp. and Bradyrhizobium spp. Inspecific embodiments, the Rhizobia cell is a Rhizobium leguminosarumcell and may further be a cell of R. leguminosarum bv. trifolii, R.leguminosarum bv. phaseoli or Rhizobium leguminosarum. bv. viciae.

In another aspect of a transformation method provided by the invention,a plant cell that is transformed may be comprised in an explant from aplant seed, for example, from a seedling, callus, cell suspension,cotyledon, meristem, leaf, root, or stem. The explant may comprise anembryonic meristem explant; callus; cell suspension; cotyledon; ortissue from leaves, roots, or stems.

A bacterium used for transformation in accordance with the invention maycomprise nucleic acids introduced, for example, by electroporation. Thesequences may comprise nucleic acid required for conjugative transferindependent of VirD2 function. The nucleic acids may include first andsecond nucleic acids.

In another aspect of the invention, a transformation method providedherein may comprise selecting a plant cell transformed with a nucleicacid of interest in the absence of a selection agent. Selecting a plantcell transformed with a nucleic acid of interest may comprise culturingthe plant cell in the presence of a selection agent, wherein the nucleicacid of interest confers tolerance to the selection agent or is operablylinked to a further nucleic acid that confers tolerance to the selectionagent. Examples of such selection agents include glyphosate, kanamycin,bialaphos or dicamba. In one embodiment, the nucleic acid of interest orfurther nucleic acid encodes EPSP synthase and in a still furtherembodiment encodes the EPSP synthase protein CP4. In another embodiment,the selection agent is glyphosate. In yet other embodiments, thesequence of interest may be defined as not physically linked to aselectable marker gene. For example, the marker gene and nucleic acid ofinterest may genetically segregate in progeny of a plant regeneratedfrom the plant cell transformed with the nucleic acid of interest.

A bacterium in accordance with the invention may comprise at least athird nucleic acid comprising a further nucleic acid of interest,wherein the plant cell is transformed with the third nucleic acid. In amethod of the invention, a plant may be regenerated a transgenic plantcell, wherein the plant comprises the sequence of interest. Regeneratinga plant may comprise inducing formation of one or more shoots from anexplant comprising the plant cell and cultivating at least a first shootinto a whole fertile plant. In certain embodiments, the plant may be acorn or cotton plant. In further embodiments, regeneration occurs byorganogenesis. In other embodiments, the plant is a soybean or canolaplant.

In another aspect, the invention provides a Rhizobia cell selected fromthe group consisting of: Rhizobium spp., Sinorhizobium spp.,Mesorhizobium spp., Phyllobacterium spp. Ochrobactrum spp. andBradyrhizobium spp., the cell comprising (i) a first nucleic acidcomprising a vir gene region of a Ti plasmid wherein the vir gene regionacts to introduce a nucleic acid coding for a sequence of interest intoa plant cell in a VirD2-dependent manner; and (ii) a second nucleic acidcomprising one or more T-DNA border sequence(s) operably linked to anucleic acid coding for a sequence of interest. In one embodiment, thecell is further defined as comprising a selectable marker. In anotherembodiment, the Rhizobia cell is selected from the group consisting of:Rhizobium sp., Rhizobium sp. NGR234, Rhizobium leguminosarum Madison, R.leguminosarum USDA2370, R. leguminosarum USDA2408, R. leguminosarumUSDA2668, R. leguminosarum 2370G, R. leguminosarum 2370LBA, R.leguminosarum 2048G, R. leguminosarum 2048LBA, R. leguminosarum bv.phaseoli, R. leguminosarum bv. phaseoli 2668G, R. leguminosarum bv.phaseoli 2668LBA, R. leguminosarum RL542C, R. leguminosarum bv. viciae,R. leguminosarum bv. trifolii, Rhizobium etli USDA 9032, R. etli bv.phaseoli, Rhizobium tropici, Mesorhizobium sp., Mesorhizobium lotiML542G, M. loti ML4404, Sinorhizobium sp., Sinorhizobium meliloti SD630,S. meliloti USDA1002, Sinorhizobium fredii USDA205, S. fredii SF542G, S.fredii SF4404, S. fredii SM542C, Bradyrhizobium sp., Bradyrhizobiumjaponicum USDA 6, and B. japonicum USDA 110. In specific embodiments,the cell is a Rhizobium leguminosarum cell and may further be. Forexample, a R. leguminosarum bv. trifolii, R. leguminosarum bv. phaseolior Rhizobium leguminosarum. bv. viciae cell.

In yet another aspect of the invention, a DNA construct is providedcompetent for virD2-independent transfer from Rhizobia and lacking T-DNAborder sequence, the construct comprising an oriT sequence and traA ormob sequence operably linked to a nucleic acid of interest. Theinvention further provides a Rhizobia cell transformed with such a DNAconstruct, wherein the Rhizobia is selected from the group consistingof: Rhizobium spp., Sinorhizobium spp., Mesorhizobium spp.,Phyllobacterium spp. Ochrobactrum spp. and Bradyrhizobium spp. In oneembodiment, the Rhizobia cell is selected from the group consisting of:Rhizobium sp., Rhizobium sp. NGR234, Rhizobium leguminosarum Madison, R.leguminosarum USDA2370, R. leguminosarum USDA2408, R. leguminosarumUSDA2668, R. leguminosarum 2370G, R. leguminosarum 2370LBA, R.leguminosarum 2048G, R. leguminosarum 2048LBA, R. leguminosarum bv.phaseoli, R. leguminosarum bv. phaseoli 2668G, R. leguminosarum bv.phaseoli 2668LBA, R. leguminosarum RL542C, R. leguminosarum bv. viciae,R. leguminosarum bv. trifolii, Rhizobium etli sUSDA 9032, R. etli bv.phaseoli, Rhizobium tropici, Mesorhizobium sp., Mesorhizobium lotiML542G, M. loti ML4404, Sinorhizobium sp., Sinorhizobium meliloti SD630,S. meliloti USDA1002, Sinorhizobium fredii USDA205, S. fredii SF542G, S.fredii SF4404, S. fredii SM542C, Bradyrhizobium sp., Bradyrhizobiumjaponicum USDA 6, and B. japonicum USDA 110. In specific embodiments,the cell is a Rhizobium leguminosarum cell, and in still furtherembodiments, may be a R. leguminosarum bv. trifolii, R. leguminosarumbv. phaseoli or Rhizobium leguminosarum. bb. viciae cell.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention.

The present invention provides methods and compositions for theefficient genetic transformation of plant cells of important cropspecies by Rhizobia. The invention overcomes substantial limitations inthe art, including limited transformation efficiency and failure todescribe techniques amenable to transformation of important crop plantsby use of non-Agrobacterial strains. For example, while use of bacteriaother than Agrobacterium has been discussed for several plant varieties,transformation frequencies have been low. In the case of rice,transformation frequencies of 0.6% and lower have been reported, withonly one transformed plant obtained from 687 inoculated calli(Broothaerts et al., 2005). This contrasts to 50-80% transformationfrequencies using Agrobacterium. Even using model organisms easilytransformed by Agrobacterium, transformation frequencies were only afraction of those obtained by Agrobacterium-mediated transformation.

To date considerable research had been required in many instances toapply even well developed transformation procedures such asAgrobacterium-mediated transformation to different plant species. Plantsof different species often exhibit substantial physiological differencesthat effect amenability to genetic transformation. Methods fortransformation of one species of plant therefore often do not workeffectively, if at all, with other plants and the ability to transform aplant is not necessarily predictive of the ability to transform evenrelated species using that procedure. This is particularly true forbacterial transformation, which involves complex biochemicalinteractions between the bacterial strains used and target plant cells.Rhizobia interact with plants in the native environment and thereforecan exhibit host-specificities, the impact of which is unknown for manycrop species.

Thus, identifying plants amenable to Rhizobia-mediated transformation,and developing procedures allowing increased transformation efficienciesis of great interest. Efficient transformation in particular isimportant because the extent to which any given transformation event isexpressed can vary substantially depending upon the integration site inthe plant genome. The ability to select transformation events having asuitable expression profile is thus dependent upon the ability toefficiently produce transformants. As explained in the working examplesbelow, transient transformation frequencies of as high as 5% wereobtained by the inventors for the transformation of soybeans (FIG. 4)and frequencies approaching 56% and 33% were obtained in the case ofcotton (FIG. 12) and canola (FIG. 9), respectively.

The present invention overcomes limitations in the art by providing, inone embodiment, techniques for the use of Rhizobia to transformimportant crop plants that were not previously known to be transformableby Rhizobia, including canola, corn, cotton, and soybean. The inventionalso provides techniques for the efficient transformation of plantsusing Rhizobia, including those already known to be amenable totransformation by Rhizobia at a low frequency. The invention alsoprovides methods for the transformation of tissue targets differing fromthose of Agrobacterium. For example, while Agrobacterium typicallyrequires a wound site to infect plants, some other members of theRhizobiales, including Rhizobiaceae such as Rhizobium, naturally infectplant roots via infection threads that penetrate plant tissues, allowingfor use of non-wounded tissue as a transformation target.

The following definitions are provided in order to aid those skilled inthe art in understanding the detailed description of the presentinvention.

As used herein, “plant growth regulator” or “plant hormone” refers tocompounds that affect plant growth. Plant growth regulators include, butare not limited to, auxins, cytokinins, ABA, gibberellins, ethylene,brassinosteroids, and polyamines. Auxins affect the elongation of shootsand roots at low concentration but inhibit growth at higher levels.Commonly used auxins include picloram (4-amino-3,5,6-trichloropicolinicacid), 2,4-D (2,4-dichlorophenoxyacetic acid), IAA (indole-3-aceticacid), NAA (α-naphthaleneacetic acid), and dicamba (3,6-dichloroanisicacid). Cytokinins cause cell division, cell differentiation, and shootdifferentiation. Commonly used cytokinins include kinetin, BA(6-benzylaminopurine), 2-ip (2-isopentenyladenine), BAP(6-benzylaminopurine), thidiazuron (TDZ), zeatin riboside, and zeatin.

“Coding sequence”, “coding region” or “open reading frame” refers to aregion of continuous sequential nucleic acid triplets encoding aprotein, polypeptide, or peptide sequence.

“Dicot” or “dicotyledonous” refers to plants having two cotyledons.Examples include, without limitation, plants such as alfalfa, beans,broccoli, cabbage, canola, carrot, cauliflower, celery, cotton,cucumber, eggplant, lettuce, melon, pea, pepper, potato, pumpkin,radish, rapeseed, spinach, soybean, squash, tomato, and watermelon.

“Endogenous” refers to materials originating from within the organism orcell.

“Exogenous” refers to materials originating from outside of the organismor cell. As used herein, exogenous is intended to refer to any nucleicacid from a source other than the recipient cell or tissue, regardlessof whether a similar (but not identical) nucleic acid may already bepresent in the recipient cell or tissue.

“Explant” refers to a plant part that is capable of being transformedand subsequently regenerated into a transgenic plant. Examples includeembryos, callus, cell suspensions, cotyledons, meristems, seedlings,seeds, leaves, stems or roots.

“Monocot” or “monocotyledonous” refers to plants having a singlecotyledon. Examples include, without limitation, onions, corn, rice,sorghum, wheat, rye, millet, sugarcane, oat, triticale, barley andturfgrass.

“Nucleic acid” refers to deoxyribonucleic acid (DNA) or ribonucleic acid(RNA).

“Phenotype” refers to a trait exhibited by an organism resulting fromthe expression (or lack of expression) of nucleic acids in the genome(including non-genomic DNA and RNA such as plasmids and artificialchromosomes) and/or organelles of the organism.

The term “plant” encompasses any higher plant and progeny thereof,including monocots (e.g., corn, rice, wheat, barley, etc.), dicots(e.g., soybean, cotton, tomato, potato, Arabidopsis, tobacco, etc.),gymnosperms (pines, firs, cedars, etc) and includes parts of plants,including reproductive units of a plant (e.g., seeds, bulbs, tubers,meristematic tissues, or other parts or tissues from that the plant canbe reproduced), fruits and flowers.

“Polyadenylation signal” or “polyA signal” refers to a nucleic acidsequence located 3′ to a coding region that promotes the addition ofadenylate nucleotides to the 3′ end of an mRNA transcribed from thecoding region.

“Promoter” or “promoter region” refers to a nucleic acid sequence,usually found 5′ to a coding sequence, that alters expression of thecoding sequence by providing a recognition site for RNA polymeraseand/or other recognition sites for other transcription-related factorsutilized to produce RNA and/or initiate transcription at the correctsite on the DNA.

“Recombinant nucleic acid vector” or “vector” or “construct” refers toany agent such as a plasmid, cosmid, virus, autonomously replicatingsequence, phage, or linear or circular single- or double-stranded DNA orRNA nucleotide segment, derived from any source, capable of genomicintegration or autonomous replication, comprising a nucleic acidmolecule in which one or more nucleic acid sequences have been linked ina functionally operative manner. Such recombinant nucleic acid vectorsor constructs typically comprise a 5′ regulatory sequence or promoterregion and a coding sequence encoding for a desired gene product. Thevectors are typically designed such that once delivered into a cell ortissue, the coding sequence is transcribed into mRNA, which isoptionally translated into a polypeptide or protein.

“Regeneration” refers to the process of growing a plant from a plantcell or tissue.

“Rhizobia” refers without limitation to bacterial genera, species, andstrains that may be assigned to the order Rhizobiales other thanAgrobacterium bacterial strains comprising the taxonomic familiesRhizobiaceae (e.g. Rhizobium spp., Sinorhizobium spp.);Phyllobacteriaceae (e.g. Mesorhizobium spp., Phyllobacterium spp.);Brucellaceae (e.g. Ochrobactrum spp.); Bradyrhizobiaceae (e.g.Bradyrhizobium spp.), and Xanthobacteraceae (e.g. Azorhizobium spp.),among others. For the purposes of the present application, “Rhizobia”does not include, biovars, or species.

Taxonomic assignment may be done as is known in the art, for instance bycomparison of 16S rDNA sequences or other classification methods. Wildtype strains of many Rhizobium species are typically able to induceformation of nitrogen fixing nodules in root tissues of host plants suchas leguminous plants (Fabaceae). However, the ability to nodulate rootsof a given plant species is not required for Rhizobium-mediated DNAtransfer into cells of the plant species.

“Selectable marker” or “screenable marker” refers to a nucleic acidsequence whose expression confers a phenotype facilitatingidentification of cells, tissues, or plants containing the nucleic acidsequence.

“Transcription” refers to the process of producing an RNA copy from aDNA template.

“Transformation” refers to a process of introducing an exogenous nucleicacid sequence into a cell or tissue. The transformation may be transientor stable. In stable transformations, part or all of the exogenousnucleic acid is incorporated (e.g., integrated or stably maintained) inthe nuclear genomic DNA, plastid DNA, or is capable of autonomousreplication in the nucleus or plastid.

“Transgenic” refers to organisms into which an exogenous nucleic acidsequence has been stably transformed.

In designing a vector for the transformation process, one or moregenetic components are selected that will be introduced into the plantcell or tissue. Genetic components can include any nucleic acid that isintroduced into a plant cell or tissue using the method according to theinvention. Genetic components can include non-plant DNA, plant DNA orsynthetic DNA.

In one embodiment, the genetic components are incorporated into a DNAcomposition such as a recombinant, double-stranded plasmid or vectormolecule comprising at least one or more of following types of geneticcomponents: (a) a promoter that functions in plant cells to cause theproduction of an RNA sequence, (b) a structural DNA sequence that causesthe production of an RNA sequence that encodes a product of agronomicutility, and (c) a 3′ non-translated DNA sequence that functions inplant cells to cause the addition of polyadenylated nucleotides to the3′ end of the RNA sequence.

The vector may contain a number of genetic components to facilitatetransformation of the plant cell or tissue and to regulate expression ofthe structural nucleic acid sequence. In one preferred embodiment, thegenetic components are oriented so as to express a mRNA, that in anoptional embodiment can be translated into a protein. The expression ofa plant structural coding sequence (a gene, cDNA, synthetic DNA, orother DNA) that exists in double-stranded form involves transcription ofmessenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzymeand subsequent processing of the mRNA primary transcript inside thenucleus. This processing involves a 3′ non-translated region that addspolyadenylated nucleotides to the 3′ ends of the mRNA.

Means for preparing plasmids or vectors containing the desired geneticcomponents are well known in the art. Vectors typically consist of anumber of genetic components, including but not limited to regulatoryelements such as promoters, leaders, introns, and terminator sequences.Regulatory elements are also referred to as cis- or trans-regulatoryelements, depending on the proximity of the element to the sequences orgene(s) they control.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the “promoter”. The promoter region contains a sequenceof bases that signals RNA polymerase to associate with the DNA and toinitiate the transcription into mRNA using one of the DNA strands as atemplate to make a corresponding complementary strand of RNA.

A number of promoters that are active in plant cells have been describedin the literature. Such promoters would include but are not limited tothe nopaline synthase (NOS) and octopine synthase (OCS) promoters thatare carried on Ti plasmids of Agrobacterium tumefaciens, thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Sand 35S promoters and the Figwort mosaic virus (FMV) 35S promoter, andthe enhanced CaMV35S promoter (e35S). A variety of other plant genepromoters that are regulated in response to environmental, hormonal,chemical, and/or developmental signals, also can be used for expressionof any DNA construct in plant cells, including, for instance, promotersregulated by (1) heat (Callis et al., 1988, (2) light (e.g., pea RbcS-3Apromoter, Kuhlemeier et al., (1989); maize RbcS promoter, Schaffner etal., (1991); (3) hormones, such as abscisic acid (Marcotte et al., 1989,(4) wounding (e.g., Wuni, Siebertz et al., 1989); or other signals orchemicals. Tissue specific expression is also known. As described below,it is preferred that the particular promoter selected should be capableof causing sufficient expression to result in the production of aneffective amount of the gene product of interest. Examples describingsuch promoters include without limitation U.S. Pat. No. 6,437,217 (maizeRS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter, OsAct1),U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362(maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S.Pat. No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos.5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat.No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (riceactin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No.5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (lightinducible promoters), U.S. Pat. No. 6,140,078 (salt induciblepromoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S.Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S.Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. Pat. No. 7,151,204(maize chloroplast aldolase promoter). Additional promoters that mayfind use are a nopaline synthase (NOS) promoter (Ebert et al., 1987),the octopine synthase (OCS) promoter (which is carried on tumor-inducingplasmids of Agrobacterium tumefaciens), the caulimovirus promoters suchas the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al.,1987), the CaMV 35S promoter (Odell et al., 1985), the figwort mosaicvirus 35S-promoter (Walker et al., 1987; U.S. Pat. Nos. 6,051,753;5,378,619), the sucrose synthase promoter (Yang et al., 1990), the Rgene complex promoter (Chandler et al., 1989), and the chlorophyll a/bbinding protein gene promoter, PClSV (U.S. Pat. No. 5,850,019). In thepresent invention, CaMV35S with enhancer sequences (e35S; U.S. Pat. Nos.5,322,938; 5,352,605; 5,359,142; and 5,530,196), FMV35S (U.S. Pat. Nos.6,051,753; 5,378,619), peanut chlorotic streak caulimovirus (PClSV; U.S.Pat. No. 5,850,019), At.Act 7 (Accession # U27811), At.ANT1 (U.S. PatentApplication 20060236420), FMV.35S-EF1a (U.S. Patent ApplicationPublication 2005/0022261), eIF4A10 (Accession # X79008) and AGRtu.nos(GenBank Accession V00087; Depicker et al, 1982; Bevan et al., 1983),rice cytosolic triose phosphate isomerase (OsTPI; U.S. Pat. No.7,132,528), and rice actin 15 gene (OsAct15; U.S. Patent ApplicationPublication 2006/0162010 promoters may be of particular benefit. In someinstances, e.g. OsTPI and OsAct 15, a promoter may include a 5′UTRand/or a first intron.

Promoter hybrids can also be constructed to enhance transcriptionalactivity (U.S. Pat. No. 5,106,739), or to combine desiredtranscriptional activity, inducibility and tissue specificity ordevelopmental specificity. Promoters that function in plants include butare not limited to promoters that are inducible, viral, synthetic,constitutive as described, and temporally regulated, spatiallyregulated, and spatio-temporally regulated. Other promoters that aretissue-enhanced, tissue-specific, or developmentally regulated are alsoknown in the art and envisioned to have utility in the practice of thisinvention.

The promoters used in the DNA constructs (i.e. chimeric/recombinantplant genes) of the present invention may be modified, if desired, toaffect their control characteristics. Promoters can be derived by meansof ligation with operator regions, random or controlled mutagenesis,etc. Furthermore, the promoters may be altered to contain multiple“enhancer sequences” to assist in elevating gene expression.

The mRNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene and can bespecifically modified so as to increase or decrease translation of themRNA. The 5′ non-translated regions can also be obtained from viralRNAs, from suitable eukaryotic genes, or from a synthetic gene sequence.Such “enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA. The present invention isnot limited to constructs wherein the non-translated region is derivedfrom both the 5′ non-translated sequence that accompanies the promotersequence. Rather, the non-translated leader sequence can be derived fromunrelated promoters or genes (see, for example U.S. Pat. No. 5,362,865).Examples of non-translation leader sequences include maize and petuniaheat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus coatprotein leaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122),PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and Freed,1990), OsAct1 (U.S. Pat. No. 5,641,876), OsTPI (U.S. Pat. No.7,132,528), and OsAct15 (U.S. Publication No. 20060162010), andAGRtu.nos (GenBank Accession V00087; Bevan et al., 1983). Other geneticcomponents that serve to enhance expression or affect transcription ortranslational of a gene are also envisioned as genetic components.

Intron sequences are known in the art to aid in the expression oftransgenes in monocot plant cells. Examples of introns include the cornactin intron (U.S. Pat. No. 5,641,876), the corn HSP70 intron (ZmHSP70;U.S. Pat. No. 5,859,347; U.S. Pat. No. 5,424,412), and rice TPI intron(OsTPI; U.S. Pat. No. 7,132,528) and are of benefit in practicing thisinvention.

Termination of transcription may be accomplished by a 3′ non-translatedDNA sequence operably linked to a recombinant transgene (e.g. the geneof interest, the identification sequence comprising a screenable gene,or the plant selectable marker gene). The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region (Fraley et al., 1983), is commonly used in thiscapacity. Polyadenylation molecules from a Pisum sativum RbcS2 gene(Ps.RbcS2-E9; Coruzzi et al., 1984), AGRtu.nos (Genbank AccessionE01312), E6 (Accession # U30508), rice glutelin (Okita et al., 1989),and TaHsp17 (wheat low molecular weight heat shock protein gene; GenBankAccession # X13431) in particular may be of benefit for use with theinvention.

In one embodiment, the vector contains a selectable, screenable, orscoreable marker gene. These genetic components are also referred toherein as functional genetic components, as they produce a product thatserves a function in the identification of a transformed plant, or aproduct of agronomic utility. The DNA that serves as a selection orscreening device may function in a regenerable plant tissue to produce acompound that would confer upon the plant tissue resistance to anotherwise toxic compound. A number of screenable or selectable markergenes are known in the art and can be used in the present invention.Examples of selectable markers and genes providing resistance againstthem are disclosed in Miki and McHugh, 2004. Genes of interest for useas a selectable, screenable, or scoreable marker would include but arenot limited to gus, gfp (green fluorescent protein), luciferase (LUX),genes conferring tolerance to antibiotics like kanamycin (Dekeyser etal., 1989), neomycin, kanamycin, paromomycin, G418, aminoglycosides,spectinomycin, streptomycin, hygromycin B, bleomycin, phleomycin,sulfonamides, streptothricin, chloramphenicol, methotrexate,2-deoxyglucose, betaine aldehyde, S-aminoethyl L-cysteine,4-methyltryptophan, D-xylose, D-mannose,benzyladenine-N-3-glucuronidase, genes that encode enzymes that givetolerance to herbicides like glyphosate (e.g.5-enolpyruvylshikimate-3-phosphate synthase (EPSPS): Della-Cioppa etal., 1987; U.S. Pat. No. 5,627,061; U.S. Pat. No. 5,633,435; U.S. Pat.No. 6,040,497; U.S. Pat. No. 5,094,945; WO04074443, and WO04009761;glyphosate oxidoreductase (GOX; U.S. Pat. No. 5,463,175); glyphosatedecarboxylase (WO05003362 and US Patent Application 20040177399; orglyphosate N-acetyltransferase (GAT): Castle et al., U.S. PatentPublication 20030083480), dalapon (e.g. dehI encoding2,2-dichloropropionic acid dehalogenase conferring tolerance to2,2-dichloropropionic acid (Dalapon; WO9927116)), bromoxynil(haloarylnitrilase (Bxn) for conferring tolerance to bromoxynil(WO8704181A1; U.S. Pat. No. 4,810,648; WO8900193A)), sulfonyl herbicides(e.g. acetohydroxyacid synthase or acetolactate synthase conferringtolerance to acetolactate synthase inhibitors such as sulfonylurea,imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide;(U.S. Pat. No. 6,225,105; U.S. Pat. No. 5,767,366, U.S. Pat. No.4,761,373; U.S. Pat. No. 5,633,437; U.S. Pat. No. 6,613,963; U.S. Pat.No. 5,013,659; U.S. Pat. No. 5,141,870; U.S. Pat. No. 5,378,824; U.S.Pat. No. 5,605,011)); encoding ALS, GST-II), bialaphos orphosphinothricin or derivatives (e.g. phosphinothricin acetyltransferase(bar) conferring tolerance to phosphinothricin or glufosinate (U.S. Pat.No. 5,646,024, U.S. Pat. No. 5,561,236, EP 275,957; U.S. Pat. No.5,276,268; U.S. Pat. No. 5,637,489; U.S. Pat. No. 5,273,894), atrazine(encoding GST-III), dicamba (dicamba monooxygenase (DMO); US PatentApplications 20030115626, 20030135879), or sethoxydim (modifiedacetyl-coenzyme A carboxylase for conferring tolerance tocyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop)(U.S. Pat. No. 6,414,222)), among others. Other selection procedures canalso be implemented including positive selection mechanisms (e.g. use ofthe manA gene of E. coli, allowing growth in the presence of mannose)and would still fall within the scope of the present invention (see alsoMiki and McHugh (2004)).

The present invention can be used with any suitable plant transformationplasmid or vector containing a selectable or screenable marker andassociated regulatory elements as described, along with one or morenucleic acids expressed in a manner sufficient to confer a particulardesirable trait. Examples of suitable structural genes of agronomicinterest envisioned by the present invention would include but are notlimited to genes for disease, insect, or pest tolerance, herbicidetolerance, genes for quality improvements such as yield, nutritionalenhancements, environmental or stress tolerances, or any desirablechanges in plant physiology, growth, development, morphology or plantproduct(s) including starch production (U.S. Pat. Nos. 6,538,181;6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production(U.S. Pat. Nos. 6,444,876; 6,426,447; 6,380,462), high oil production(U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; 6,476,295), modifiedfatty acid content (U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465;6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461;6,459,018), high protein production (U.S. Pat. No. 6,380,466), fruitripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition(U.S. Pat. Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; 6,171,640),biopolymers (U.S. Pat. RE37,543; U.S. Pat. Nos. 6,228,623; 5,958,745 andU.S. Patent Publication No. US20030028917). Also environmental stressresistance (U.S. Pat. No. 6,072,103), pharmaceutical peptides andsecretable peptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075;6,080,560), improved processing traits (U.S. Pat. No. 6,476,295),improved digestibility (U.S. Pat. No. 6,531,648) low raffinose (U.S.Pat. No. 6,166,292), industrial enzyme production (U.S. Pat. No.5,543,576), improved flavor (U.S. Pat. No. 6,011,199), nitrogen fixation(U.S. Pat. No. 5,229,114), hybrid seed production (U.S. Pat. No.5,689,041), fiber production (U.S. Pat. Nos. 6,576,818; 6,271,443;5,981,834; 5,869,720) and biofuel production (U.S. Pat. No. 5,998,700).Any of these or other genetic elements, methods, and transgenes may beused with the invention as will be appreciated by those of skill in theart in view of the instant disclosure.

Alternatively, the DNA sequences of interest can affect these phenotypesby the inhibition of expression of an endogenous gene via gene silencingtechnologies such cosuppression, antisense, RNAi, expression of miRNAs(natural or engineered), expression of trans-acting siRNAs, andexpression of ribozymes (see e.g., U.S. Patent Application Publication20060200878).

Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species, but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term “exogenous” isalso intended to refer to genes that are not normally present in thecell being transformed, or perhaps simply not present in the form,structure, etc., as found in the transforming DNA segment or gene, orgenes that are normally present yet that one desires, e.g., to haveover-expressed. Thus, the term “exogenous” gene or DNA is intended torefer to any gene or DNA segment that is introduced into a recipientcell, regardless of whether a similar gene may already be present insuch a cell. The type of DNA included in the exogenous DNA can includeDNA that is already present in the plant cell, DNA from another plant,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

In light of this disclosure, numerous other possible selectable orscreenable marker genes, regulatory elements, and other sequences ofinterest will be apparent to those of skill in the art. Therefore, theforegoing discussion is intended to be exemplary rather than exhaustive.

For Rhizobia-mediated transformation, after the construction of theplant transformation vector or construct, the nucleic acid molecule,prepared as a DNA composition in vitro, is introduced into a suitablehost such as E. coli and mated into another suitable host such asRhizobia, including Rhizobium, or directly transformed (e.g.electroporated) into competent Rhizobia. The Ti or Ri plasmid may benaturally transferred into nitrogen-fixing Rhizobium and may inducetumors or hairy roots, respectively (Hooykaas et al. 1977, Weller et al.2004). Such Ti or Ri plasmid may alternatively be “disarmed”, and unableto cause plant cell proliferation. Since Rhizobium and Agrobacteriumhave differing infection mechanisms, deep infection by Rhizobium orother Rhizobia through its infection thread may increase the frequencyof germ line transformation of a gene of interest during soybeantransformation once the Ti or Ri helper plasmid is introduced.

The present invention encompasses the use of bacterial strains tointroduce one or more genetic components into plants. In one embodiment,the hosts contain disarmed Ti or Ri plasmids that do not contain theoncogenes that cause tumorigenesis or rhizogenesis, derivatives of whichare used as the vectors and contain the genes of interest that aresubsequently introduced into plants. In another embodiment, the bacteriatransfer DNA into plant cells by means of a T4SS-independent mechanism,namely oriT-mediated conjugal transfer. Functions needed forT4SS-independent DNA transfer may reside on the plasmid containing theDNA to be transferred, or may reside on the chromosome or anotherplasmid, including a Ti or Ri plasmid, also present in such a bacterialcell.

Bacterial species and strains include but are not limited to Rhizobiumsp., Rhizobium sp. NGR234, Rhizobium leguminosarum Madison, R.leguminosarum USDA2370, R. leguminosarum USDA2408, R. leguminosarumUSDA2668, R. leguminosarum 2370G, R. leguminosarum 2370LBA, R.leguminosarum 2048G, R. leguminosarum 2048LBA, R. leguminosarum bv.phaseoli, R. leguminosarum bv. phaseoli 2668G, R. leguminosarum bv.phaseoli 2668LBA, R. leguminosarum RL542C, R. leguminosarum bv. viciae,R. leguminosarum bv. trifolii, Rhizobium etli USDA 9032, R. etli bvphaseoli, Rhizobium tropici, Mesorhizobium sp., Mesorhizobium lotiML542G, M. loti ML4404, Sinorhizobium sp., Sinorhizobium meliloti SD630,S. meliloti USDA1002, Sinorhizobium fredii USDA205, S. fredii SF542G, S.fredii SF4404, S. fredii SM542C, Bradyrhizobium sp., Bradyrhizobiumjaponicum USDA 6, B. japonicum USDA 110.

Any suitable plant culture medium can be used to develop or maintain aplant tissue culture, supplemented as appropriate with additional plantgrowth regulators including but not limited to auxins such as picloram(4-amino-3,5,6-trichloropicolinic acid), 2,4-D(2,4-dichlorophenoxyacetic acid) and dicamba (3,6-dichloroanisic acid);cytokinins such as BAP (6-benzylaminopurine) and kinetin; ABA; andgibberellins. Other media additives can include but are not limited toamino acids, macro elements, iron, microelements, inositol, vitamins andorganics, carbohydrates, undefined media components such as caseinhydrolysates, with or without an appropriate gelling agent such as aform of agar, such as a low melting point agarose or Gelrite if desired.Those of skill in the art are familiar with the variety of tissueculture media, which when supplemented appropriately, support planttissue growth and development and are suitable for plant transformationand regeneration. These tissue culture media can either be purchased asa commercial preparation, or custom prepared and modified. Examples ofsuch media would include but are not limited to Murashige and Skoog(1962), N6 (Chu et al., 1975), Linsmaier and Skoog (1965), Uchimiya andMurashige (1962), Gamborg's media (Gamborg et al., 1968), D medium(Duncan et al., 1985), McCown's Woody plant media (McCown and Lloyd,1981), Nitsch and Nitsch (1969), and Schenk and Hildebrandt (1972) orderivations of these media supplemented accordingly. Those of skill inthe art are aware that media and media supplements such as nutrients andgrowth regulators for use in transformation and regeneration and otherculture conditions such as light intensity during incubation, pH, andincubation temperatures that can be optimized for the particular varietyof interest.

After a transformable plant tissue is isolated or developed in tissueculture, or transformable plant tissue is identified and/or prepared inplanta, the next step of the method is introducing the geneticcomponents into the plant tissue. This process is also referred toherein as “transformation.” The plant cells are transformed andoptionally subject to a selection step. The independent transformantsare referred to as transgenic events. A number of methods utilizingAgrobacterium strains have been reported and can be used to insertgenetic components into transformable plant tissue. However,non-Agrobacterium spp. had not typically been utilized to transformplants.

Those of skill in the art are aware of the typical steps in the planttransformation process. The Rhizobia to be used can be prepared eitherby inoculating a liquid medium such as TY or YEM media (Beringer et al.,1974) directly from a glycerol stock or streaking the bacteria onto asolidified media from a glycerol stock, allowing the bacteria to growunder the appropriate selective conditions. The Rhizobia may be“pre-induced” by growth under nutritional or cultural conditionsincluding the presence of acetosyringone in an amount that facilitatestransformation. Those of skill in the art are familiar with proceduresfor growth and suitable culture conditions for bacteria as well assubsequent inoculation procedures. The density of the bacterial cultureused for inoculation and the ratio of the number of bacterial cells toamount of explant tissue can vary from one system to the next, andtherefore optimization of these parameters for any transformation methodis expected.

The next stage of the transformation process is the inoculation. In thisstage the suitably prepared plants, plant tissues, or explants, and thebacterial cell suspension are mixed together. The duration and conditionof the inoculation and bacterial cell density will vary depending on theplant transformation system. Growth or inoculation of transformingbacteria may occur in the presence of acetosyringone, or other knowninducer of expression of the virulence genes located on Ti or Riplasmids. In certain embodiments, growing of the bacterium other thanAgrobacterium sp. is done under conditions to minimize polysaccharideproduction during growth in induction medium. In particular embodiments,the carbon source used to minimize polysaccharide production duringRhizobia growth in induction medium is glucose in AB-TY medium, orL-arabinose and potassium gluconate in ATA medium.

After inoculation any excess bacterial suspension can be removed and thebacteria and target plant material are co-cultured. The co-culturerefers to the time post-inoculation and prior to transfer to an optionaldelay or selection medium. Any number of plant tissue culture media canbe used for the co-culture step. Plant tissues after inoculation withbacteria may be cultured in a liquid or semi-solid media. The co-cultureis typically performed for about one to four days.

After co-culture with bacteria, the inoculated plant tissues or explantscan optionally be placed directly onto selective media. Alternatively,after co-culture with bacteria, they could be placed on media withoutthe selective agent and subsequently placed onto selective media. Thoseof skill in the art are aware of the numerous modifications in selectiveregimes, media, and growth conditions that can be varied depending onthe plant system and the selective agent. Typical selective agentsinclude but are not limited to antibiotics such as geneticin (G418),kanamycin and paromomycin, or the herbicides glyphosate, glufosinate,and DICAMBA. Additional appropriate media components can be added to theselection or delay medium to inhibit bacterial growth. Such mediacomponents can include, but are not limited to, antibiotics such ascarbenicillin or cefotaxime.

The cultures are subsequently transferred to a medium suitable for therecovery of transformed plantlets. Those of skill in the art are awareof the number of methods to recover transformed plants. A variety ofmedia and transfer requirements can be implemented and optimized foreach plant system for plant transformation and recovery of transgenicplants. Consequently, such media and culture conditions disclosed in thepresent invention can be modified or substituted with nutritionallyequivalent components, or similar processes for selection and recoveryof transgenic events, and still fall within the scope of the presentinvention.

Once the transformable plant tissue is inoculated, plant cells in thetissue may be transformed, and independently transformed plant cells areselected. The independent transformants are referred to as transgenicevents. Agrobacterium-mediated transformation and regeneration systemsfor many monocot and dicot plant species are known in the art (e.g.Komari et al., 1998; Zhou et al. 1995; Hiei et al., 1994. Plant J.;6:271-282; Ishida et al. 1996; Rogers et al., 1987; Schrammeijer et al.,1990; U.S. Pat. No. 6,384,301), although use of Rhizobia for plant celltransformation has been reported only for tobacco, Arabidopsis, and rice(Broothaerts et al., 2005). Following transformation and regeneration,transgenic plants are identified. Finally, one of skill in the art willrecognize that after the expression cassette is stably incorporated intransgenic plants and confirmed to be operable, it can be introducedinto other plants by sexual crossing. Any of a number of standardbreeding techniques can be used, depending upon the species to becrossed.

The transformants produced, and their progeny, may subsequently beanalyzed to determine the presence or absence of a particular nucleicacid of interest contained on the transformation vector. Molecularanalyses can include but are not limited to Southern blots (Southern,1975), PCR (polymerase chain reaction) analyses, analysis of enzymaticactivities, immunodiagnostic approaches, and field evaluations and thelike (Also see, for example, Sambrook et al., 1989). These and otherwell known methods can be performed to confirm the stability of thetransformed plants produced by the methods disclosed.

The above-described techniques may be suitable for any plant and isespecially useful for plants such as alfalfa, barley, beans, beet,broccoli, cabbage, carrot, canola, cauliflower, celery, Chinese cabbage,corn, cotton, cucumber, dry bean, eggplant, fennel, garden beans, gourd,leek, lettuce, melon, oat, okra, onion, pea, pepper, pumpkin, peanut,potato, pumpkin, radish, rice, sorghum, soybean, spinach, squash, sweetcorn, sugarbeet, sunflower, tomato, watermelon, and wheat.

EXAMPLES

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. All references cited herein are incorporated herein byreference to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, or compositionsemployed herein.

Example 1 Rhizobium and Agrobacterium Strains

Agrobacterium tumefaciens AGL0 was obtained from ATCC (ATCC Number:BAA-100™, Lazo et al., 1991). Rhizobium leguminosarum strain Madison andSinorhizobium meliloti SD630 were isolated from weed clover in a homegarden in Madison, Wis., USA, and confirmed by sequencing the PCRproduct of a 16S rRNA amplified with the following primers: 5′GAGAGTTTGATCCTGGCTCAG 3′ (Xd578; SEQ ID NO:1) and 5′AAGGAGGTGATCCAGCCGCAG 3′ (Xd579; SEQ ID NO:2). Other Rhizobium strainswere obtained from USDA Rhizobium collection center (Table 1). Rhizobiumstrains were grown in TY or MAG medium and Agrobacterium in LB medium.Strains are shown below and 16s rRNA sequences amplified in strainsisolated are provided as SEQ ID NOs:24-30.

TABLE 1 Agrobacterium and Rhizobium strains Strain Name Ti plasmidSource A. tumefaciens AGL0 pTiBo542 ATCC; Lazo et al., 1991 A.tumefaciens LBA4404 pAL4404 Hoekema et al., 1983 A. tumefaciens AGL0CpTiBo542C (kanR) This study A. tumefaciens AGL0G pTiBo542G (kanR) Thisstudy A. tumefaciens 4404TIK pTi4404kan (kanR) This study A. tumefaciensABI pTiC58 (kanR, gentR) Monsanto Rhizobium leguminosarum None Thisstudy Madison Sinorhizobium meliloti SD630 None This study Rhizobiumleguminosarum None USDA Rhizobium collection USDA2370 center Rhizobiumleguminosarum bv. None USDA Rhizobium collection trifolii USDA2048center Rhizobium leguminosarum bv. None USDA Rhizobium collectionphaseoli USDA2668 center Sinorhizobium fredii None USDA Rhizobiumcollection USDA205 center Sinorhizobium meliloti None USDA Rhizobiumcollection USDA1002 center Mesorhizobium loti USDA None USDA Rhizobiumcollection 3471 center Bradyrhizobium japonicum None USDA Rhizobiumcollection USDA 6 center Bradyrhizobium japonicum None USDA Rhizobiumcollection USDA 110 center Rhizobium etli USDA 9032 None USDA Rhizobiumcollection (CFN42) center R. leguminosarum 2370G pTiBo542G This study R.leguminosarum 2370LBA pTi4404kan This study R. leguminosarum trifoliibv. pTiBo542G This study 2048G R. leguminosarum trifolii bv. pTi4404kanThis study 2048LBA R. leguminosarum phaseoli bv. pTiBo542G This study2668G R. leguminosarum phaseoli bv. pTi4404kan This study 2668LBA R.leguminosarum RL542C pTiBo542C This study S. fredii SF542G pTiBo542GThis study S. fredii SF4404 pTi4404kan This study S. meliloti SM542CpTiBo542C This study M. loti ML542G pTiBo542G This study M. loti ML4404pTi4404kan This study

Example 2 Transformation of Agrobacterium

The Agrobacterium competent cells were prepared by washing a log phaseculture in LB medium with chilled deionized water and 10% glycerol, andstored at −80° C. Fifty microliters of thawed competent cells were mixedwith 1 or 2 μl DNA on ice and electroporated in 1 mm gap curvet with 200ohm resistance, 25 μF capacity and 1.8 kv using a BIO-RAD Gene Pulser®II device (BIO-RAD, Hercules, Calif.).

Example 3 Construction of Ti Plasmids with an Antibiotic SelectableMarker Gene

To select Ti plasmids in Rhizobium spp., a homologous sequence wasamplified from a corresponding Ti plasmid and inserted into a kanamycinresistance vector. The homologous sequence was used to integrate thekanamycin resistance gene into the Ti plasmid by homologousrecombination.

To construct the pTiBo542C plasmid, the entire virC gene (Genbankaccession number AB027257) from the AGL0 Agrobacterium strain wasamplified with PCR using the following primers 5′ACAATAATGTGTGTTGTTAAGTCTTGTTGC 3′ (Xd683 SEQ ID NO:3) and 5′CTCAAACCTACACTCAATATTTGGTGAG 3′ (Xd684 SEQ ID NO:4) and Pfu polymerase(STRATAGENE, La Jolla, Calif.) and inserted into the TOPO cloning bluntvector (Invitrogen Carlsbad, Calif.) giving rise to an intermediatevector pMON67402 The intermediate vector was further ligated to a trfAfragment from pCGN11206 digested with PvuII/MscI, which resulted inconstruct pMON96913 (FIG. 6). The vector was then introduced into theAGL0 Agrobacterium strain by standard electroporation as outlined above,and plated on Kanamycin 50 mg/l LB medium to select for a singlecrossover event. Since the integration vector is not maintained in AGL0cells, the resistant colonies were presumably due to integration of thevector into a Ti plasmid by homologous recombination. The resultingstrain was designated AGL0C.

Similarly, to construct the pTi542G plasmid, the entire virG sequence(Genbank accession number AB027257) from the AGL0 Agrobacterium strainwas PCR amplified using 5′ AGATCTGGCTCGCGGCGGACGCAC 3′ (Xd681; SEQ IDNO:5) and 5′ CGCTCGCGTCATTCTTTGCTGGAG 3′ (Xd682; SEQ ID NO:6) with Pfupolymerase and inserted into pMON67402, which resulted in constructpMON96026 (FIG. 8). This vector was introduced into the AGL0 strain bystandard electroporation and plated on kanamycin 50 mg/l LB medium toselect for a single crossover event. The resulting strain was designatedAGL0G.

In order to construct the pTi4404kan helper plasmid, the traI and trbCEregion of octopine Ti plasmid pAL4404 (Genbank accession numberNC_(—)002377) from LBA4404 was PCR amplified with primers 5′TCAGCAGGATGACGCCGTTATCG 3′ (Xd695; SEQ ID NO:7) and 5′TCTCGCCCGACCAATACCAACAC 3′ (Xd696; SEQ ID NO:8) (sequence from GenbankAF242881) with Pfu polymerase, and inserted into pMON67402. Theintermediate vector was further ligated to a trfA fragment frompCGN11206 digested with PvuII/MscI, which resulted in constructpMON96914 (FIG. 7). This plasmid vector was introduced into LBA4404 byelectroporation and selected on LB medium with kanamycin 50 mg/l toselect for a single crossover event.

After a three day culture on solid medium, the kanR resistant colonieswere transferred into 2 mls of liquid LB medium with kanamycin 50 mg/l.One microliter of overnight culture was directly amplified withYieldAce® Taq polymerase following manufacturer instruction (Stratagene)with the following primers: 5′ GCTGACGGGCCCGGATGAATGTCAGCTACTG 3′(Xd715; SEQ ID NO:9) and 5′ GCTCTAGAAATTGTAAGCGTTAATAATTCAGAAGAACTCGTC3′ (Xd716; SEQ ID NO:10) and integration of the kanamycin resistancegene into Ti plasmids was confirmed. The resulting strain was designatedas 4404TIK.

Example 4 Extraction of Ti Plasmids from Agrobacterium

The modified Ti plasmids, pTiBo542C, pTiBo542G, pTi4404kan and pTiC58(ABI), were extracted from the modified Agrobacterium strains AGL0C,AGL0G, 4404TIK and ABI, containing the respective plasmids. Five mls ofovernight culture in LB with kanamycin 50 mg/l was spun down,resuspended in 400 μl of P1 buffer, mixed with 400 μl of P2 buffer,neutralized with 400 μl P3 buffer (buffers from QIAGEN maxi-prep kit).After 5 min incubation at room temperature, the mixture was spun for 10min at 12 g at 4° C. Approximately 1200 μl of supernatant was mixed with800 μl of isopropanol and spun for 10 min at 4° C. The pellet was washedwith 70% ethanol once and resuspended in 200 μl of TE without drying.The mega plasmids were subsequently stored at 4° C.

Example 5 Rhizobia Competent Cells and Introduction of Modified TiPlasmids into Rhizobia

Rhizobia competent cells were prepared according to Garg et al. 1999with modification. Briefly, one loopful of Rhizobia (e.g. Rhizobium)cells from a frozen glycerol stock was grown in 10 mls of TY broth for24 hours, and then transferred into 500 ml TY broth at 30° C. withvigorous shaking and allowed to grow to mid-logarithmic phase(OD₆₀₀=0.4-0.6). The culture was transferred into two 250 centrifugetubes, chilled on ice for 15-30 min and centrifuged at 9,000 rpm for 10min at 4° C. to harvest cells. The cell pellet was washed with coldsterile deionized water, and with 10% cold glycerol and resuspended in10% cold glycerol. The cell suspension was aliquoted at 50 μl/tube forimmediate use or frozen in liquid nitrogen and stored at −80° C.

Electroporation of the modified Ti plasmids into Rhizobia strains: Fiftymicroliters of the competent cells were thawed on ice, mixed with 1 or 2μls of the prepared Ti plasmid, and kept on ice for 30 min. The mixturewas transferred into a chilled 1 mm-gap electroporation cuvette. Theelectroporation parameters (BIO-RAD Gene Pulser® II) were set asfollows: 2 KV/400Ω resistance/25 μF capacity or 1.5 KV/400Ωresistance/25 μF capacity or 1.5 KV/800Ω resistance/10 μF capacity.After electroporation, the cuvette was kept on ice for 5-10 min beforeadding 1 ml of TY or MAG medium and transferring into a 14-ml Falcontube. The tube was cultured for 3 hours at 30° C., plated onto TY or MAGsolid medium with 50 mg/l kanamycin and cultured at 30° C. for threedays to recover resistant colonies.

Confirmation of Rhizobia transformed with Ti plasmids: The kanamycinresistant colonies were transferred into 3 mls of liquid TY or MAGmedium with 50 mg/l kanamycin and cultured overnight. One microliter ofculture was directly amplified with YieldAce® Taq polymerase followingmanufacturer's instructions (Stratagene).

To detect pTiBo542C or pTiBo542G in Rhizobia strains, the virC primers5′ ACAATAATGTGTGTTGTTAAGTCTTGTTGC 3′ (Xd683; SEQ ID NO:3) and 5′CAATTGCATTTGGCTCTTAATTATCTGG 3′ (Xd684a; SEQ ID NO:11) or virG primers5′ AGATCTGGCTCGCGGCGGACGCAC 3′ (Xd681; SEQ ID NO:5) and 5′CGCTCGCGTCATTCTTTGCTGGAG 3′ (Xd682; SEQ ID NO:6) were used to amplify a2.35 kb or a 1.2 kb fragment, respectively.

For the pTi4404kan plasmid, the following primers were used: 5′GCATGCCCGATCGCGCTCAAGTAATC 3′ (Xd699; SEQ ID NO:12) and 5′TCTAGGTCCCCCCGCGCCCATCG 3′ (Xd700; SEQ ID NO:13)) amplifies a 1274 bpvirD2 coding sequence for the octopine Ti plasmid; 5′CCATGGATCTTTCTGGCAATGAGAAATC 3′ (Xd701; SEQ ID NO:14) and 5′GTCAAAAGCTGTTGACGCTTTGGCTACG 3′ (Xd702: SEQ ID NO:15) amplifies a 1602bp virE2 fragment; 5′ ACGGGAGAGGCGGTGTTAGTTGC 3′ (Xd703; SEQ ID NO:16)and 5′ CGATAGCGACAATGCCGAGAACG 3′ (Xd704; SEQ ID NO:17) amplifiesapproximately a 0.9 kb virB1 fragment.

In order to identify the pTiC58 plasmid from the ABI strain, three pairsof primers were used: 5′ ATGCCCGATCGAGCTCAAGTTATC 3′ (Xd685; SEQ IDNO:18) and 5′ TGAAAGGACACCTCTCCGTTGCTG 3′ (Xd686; SEQ ID NO:19)amplifies a 1247 bp virD2 fragment; 5′ CCATGGATCCGAAGGCCGAAGGCAATG 3′(Xd687; SEQ ID NO:20) and 5′ CTACAGACTGTTTACGGTTGGGC 3′ (Xd688; SEQ IDNO:21) amplifies a 1670 bp virE2 entire coding sequence; 5′GTGAGCAAAGCCGCTGCCATATC 3′ (Xd689; SEQ ID NO:22) and 5′TAGAGCGTCTGCTTGGTTAAACC 3′ (Xd690; SEQ ID NO:23) amplifies a 1102 bppartial repA fragment.

Example 6 Media for Bacterial Growth

Media used for Rhizobia growth in the Rhizobia-mediated transformationprotocol employed to develop transformed plants were prepared usingstandard methods known to one skilled in the art. Media formulations areas follows:

TY medium per L Bactotryptone   5 g/L Yeast extract   3 g/L CaCl₂•2H₂O0.87 g/L pH 7.0 For solid TY medium, add 15 g/l Bacto-Agar beforeautoclaving.

MAG Medium Liquid (From USDA Rhizobium collection center) HEPES 1.3 g/LMES 1.1 g/L Yeast extract 1 g/L L-Arabinose 1 g/L Potassium gluconate 1g/L KH₂PO₄ 0.22 g/L Na₂SO₄ 0.25 g/L pH 6.6 with KOH Autoclave and addthe following filter sterilized stock solution: NH₄Cl (16 g/100 ml) 2ml/l FeCl₃ (0.67 g/100 ml, FS) 1.0 ml/l CaCl₂ (1.5 g/100 ml) 1.0 ml/lMgSO₄ (18 g/100 ml) 1.0 ml NaMoO₄•2H₂O (1 g/100 ml) 1.0 ml/l NiCl₂•6H₂O(2.2 g/100 ml) 0.1 ml/l For solid MAG medium, add 15 g/l Bacto-Agarbefore autoclaving. LB medium Per liter Bacto-tryptone 10 g Bacto-yeastextract 5 g NaCl 10 g Adjust pH to 7.5 with sodium hydroxide. Afterautoclaving, distribute into culture plate (25 ml/plate) For solid LBmedium, add 15 g/l Bacto-Agar before autoclaving AB Minimal Medium: 20×AB Buffer: K₂HPO₄ 60 g/l NaH₂PO₄ 20 g/l Autoclave separately 20× ABSalts (Filter sterilized, keep in dark): NH₄Cl 20 g/l MgSO₄•7H₂O 6 g/lKCl 3 g/l CaCl₂ 0.2 g/l FeSO₄•7H₂O 50 mg/l pH to 7 before autoclavingCombine 50 ml AB Buffer and 50 ml AB Salts with 900 ml sucrose- water(final concentration of sucrose in one liter is 0.5%). 1× AB-TYInduction medium: Glucose 5 g 20× AB buffer 50 ml 20× AB Salt stock 50ml TY medium 20 ml Add sterile water to 1000 ml Adjust to pH 5.4 with100 mM MES Add 100 μM acetosyringone (1 M stock in DMSO; add 1 μlstock/10 ml medium after suspending bacteria)ATA Medium for vir Induction in Rhizobium:The ATA (AB minimal medium+TY+Arabinose) medium was modified from AB-TYmedium using arabinose and potassium gluconate to replace glucose. Thegrowth rate of all Rhizobia is almost doubled in this medium. Thebacteria produced much less polysaccharide in this medium and thebacterial pellets were much tighter.

L-Arabinose 1 g/L Potassium gluconate 1 g/L 20× AB buffer 50 ml 20× ABsalt stock 50 ml TY medium 20 ml Add sterile water to 1000 ml pH 5.4with 100 mM MES Add 200 μM acetosyringone (1 M stock in DMSO) afterresuspending bacteria.

Example 7 Crop Transformation Vectors for Use with Modified RhizobiaStrains

Rhizobium transformation vectors were constructed using standardmolecular techniques known to those skilled in the art. Plasmidconstructs pMON96033 (FIG. 1; for soybean and canola transformation),pMON96036 (FIG. 2; for corn transformation), or pMON101316 (FIG. 3; forcotton transformation) were employed. All three constructs contain apVS1 replication origin, and either GUS, GFP, or both reporter genes.Recombinant plasmids were transferred into various modified Rhizobiastrains by electroporation and confirmed by restriction enzyme digestionof miniprep DNA.

The FMV CP4 gene used in constructing the plasmids has a promoter fromFigwort Mosaic Virus (FMV) followed by the CP4syn gene, a synthetic geneencoding CP4 EPSP synthase. See, U.S. Pat. No. 5,633,435, which isincorporated by reference herein. EPSP synthase, when expressed, confersa substantial degree of glyphosate resistance upon the plant cell andplants generated there from. The e35s GUS gene is a β-glucuronidasegene, which is typically used as a histochemical marker, behind the e35Spromoter. The FMV GUS gene is the FMV promoter with GUS. The NOS NPTIIgene has a neomycin phosphotransferase gene, which confers resistance tokanamycin, behind the promoter for the nopaline synthase gene (NOS). TheAct 1 GFP gene has an actin promoter from rice and the gene for greenfluorescence protein, which is a screenable marker. The e35s GFP gene isthe gene for green fluorescence protein behind the e35S promoter.Overnight cultures of a Rhizobia strain containing the plasmid used weregrown to log phase and then diluted to a final optical density of 0.3 to0.6.

Example 8 Rhizobia-Mediated Soybean Transformation

Transformation was performed using an organogenesis process, asdescribed by Martinell et al. (U.S. Pat. No. 7,002,058), withmodifications. pMON96033 containing the GUS and CP4 genes weretransferred into various modified Rhizobia strains (e.g. Rhizobium sp.,Mesorhizobium sp., Sinorhizobium sp.) by electroporation. Singlecolonies were recovered on MAG or TY medium with 50 mg/l spectinomycinand 50 mg/l kanamycin and inoculated in 20-50 mls of liquid TY mediumwith the same selection in a shaker at 30° C. at 200 rpm. The presenceof plasmid in the Rhizobia culture was verified by restriction enzymedigestion of mini-prepared plasmid from 10 ml culture. The remainingliquid culture was mixed with glycerol to a final concentration of 20%,aliquoted and stored at −80° C. as seed cultures.

To prepare Rhizobia inoculum, 0.25-1 ml frozen seed culture wasinoculated into 250 or 500 mls of TY medium with the same antibioticselection as above and grown overnight at 28° C. with shaking at 200 rpmto mid-log growth phase. The culture was spun down and directlysuspended in an inoculation medium (INO medium) at the concentration ofOD₆₆₀ about 0.3.

Induced Rhizobia culture was also used in soybean transformation. Toinduce the Rhizobia, the overnight culture was resuspended in AB-TYmedium at an OD₆₆₀ of about 0.3 and acetosyringone was added to a finalconcentration of 100 μM. The culture was further shaken overnight at 28°C., spun down and re-suspended in the inoculation medium (INO medium) toa concentration of OD₆₆₀ about 0.3.

Soybean cultivar A3525 (U.S. Pat. No. 7,002,058) was used forRhizobia-mediated transformation. The method was modified forRhizobia-mediated transformation as follows. Soybean seeds weregerminated at room temperature in BGM medium and meristem explants fromsoy mature seeds were excised by machine (U.S. Application 20050005321).Soybean meristem explants in a PLANTCON lid were mixed with Rhizobiasuspension in INO medium and sonicated in a W-113 Sonicator (HondaElectronics Co., Ltd, Aichi, Japan). After sonication, the explants wereco-cultured in the same PLANTCON for 1-11 days at 23° C. with a 16/8hour light-dark photo period. The explants were then transferred ontothe surface of the WPM selection medium containing 75 μM glyphosate.After 2 weeks, explants were transferred again to 75 μM glyphosate solidWPM medium. Shoots with fully expanded trifolia were recovered after6-10 weeks post-inoculation and rooted in BRM medium (optionally withfungicide) containing 0.1 mg/l IAA and 25 μM glyphosate selection. Therooted plantlets were transferred to the greenhouse for maturity.

TABLE 2 Media components for soy transformation. amount/L Compound BGMmedium for soybean seed germination 0.505 g Potassium nitrate 0.24 gAmmonium nitrate 0.493 g Magnesium sulfate 0.176 g Calcium chloride 27.2mg Potassium phosphate monobasic 1.86 mg Boric acid 5.07 mg Manganesesulfate 2.58 mg Zinc sulphate 0.249 mg Potassium iodide 0.216 mg SodiumMolybdate 0.0008 mg Copper sulphate 0.0008 mg Cobalt chloride stock 3.36mg Disodium EDTA 2.49 mg Ferrous sulphate 1.34 mg Thiamine HCl 0.5 mgNicotinic acid 0.82 mg Pyridoxine HCl 20 g/L Sucrose (Ultra Pure) 125 mgCefotaxime pH 5.6 INO medium for soy co-culture 1/10× of Gamborg B5medium micronutrient and vitamin components; 2/5× of macronutrients 1 gPotassium Nitrate (KNO₃) 30 g Glucose 3.9 g MES (pH 5.4) Afterautoclaving, lipoic acid added to inoculum to a final concentration of250 μM SOY WPM shooting medium amount/L Compound 2.41 g WPM Powder(PhytoTech Laboratories) 20 g Sucrose (Ultra Pure) 1.29 g CalciumGluconate (Sigma) 4.0 g AgarGel (pH 5.6) mL/L Post-autoclavingingredients 4 mL Cefotaxime (50 mg/mL) 1 ml Ticarcillin (100 mg/ml) 5 mLCarbenicillin (40 mg/mL) 0.15 mL Glyphosate (0.5 FS Stock) (0.075 mM)BRM rooting medium amount/L Compound 2.15 g MS Powder (Phytotech) 0.1 gmyo-Inositol 2 mg Glycine 0.5 mg Nicotinic acid 0.5 mg Pyridoxine HCl0.1 mg Thiamine HCl 30 g Sucrose (Ultra Pure) 10 ml L-Cysteine (10mg/ml) 8 g Washed Agar mL/L Post-autoclaving ingredients 5.0 IAA (0.033mg/ml in 1 mM KOH) 1 mL Ticarcillin (100 mg/ml) 0.05 mL Glyphosate (0.5FS Stock) (0.025 mM)

The binary vector pMON96033 was transferred into Rhizobia strains andco-cultivated with soybean meristem explants, and GUS positive resultswere observed (Table 3 and FIG. 4). S. meliloti, S. fredii, M. loti andone R. leguminosarum showed T-DNA delivery into soybean explantsdemonstrated by small blue spots of GUS activity. Transgenic soybeanplants were obtained from Rhizobia-mediated transformation experimentswith various strains (Table 4). The transgenic nature of these soybeanplants were confirmed by transgene copy number assay, where most of thetransformants revealed 1-2 copy simple integration pattern. (Table 5).

TABLE 3 Transient Expression of gus Gene with Rhizobia-mediated T-DNADelivery in soybean meristem explants.* Strain Origin Transient GUSassay RL4404 Rhizobium leguminosarum strain Madison + pAL4404 + 2370LBARhizobium leguminosarum strain + USDA2370 + pTiBo542C 2370G Rhizobiumleguminosarum strain + USDA2370 + pTiBo542G SF4404 Sinorhizobium frediiUSDA205 + pAL4404 + SF542C Sinorhizobium fredii USDA205 + pTiBo542C +SM542C Mesorhizobium loti USDA3471 + pAL4404 + ML4404 Mesorhizobium lotiUSDA3471 + pAL4404 + ML542G Mesorhizobium loti USDA3471 + pTiBo542G +*Transient assays were performed after 4 day co-culture period.

TABLE 4 Rhizobia-mediated soy transformation summary. Strains SoyExplants Rooted Plants TF RL 4404 3705 2 0.05% SF 4404 5553 2 0.04% SM542C 2555 1 0.04%

TABLE 5 Copy Number Assay of Transgenic Plants from Rhizobia-mediatedTransformation* NOS copy RL4404 SF4404 SM542C 0 copy 0 0 1 1-2 copy 1 20 >2 copy 0 0 0 Total Plant 1 2 1 *Copy number was analyzed by INVADERmethod (Third Wave Technologies, Madison, WI) using a nos probe andcompared with an internal genome control.

To test if the gus transgene was transmitted to the seed progeny, seedsof two soy transgenic lines derived from Rhizobia-mediatedtransformation were stained in GUS solution (FIG. 5). The GM_A9196D linewas found to have one copy of the linked nos gene as assayed by theINVADER method. Twelve R1 seeds from this line were assayed for GUS byhistochemical staining after imbibition and removal of seed coat, and 9were GUS positive, indicating a segregation ratio of 3:1 for one copyinsert.

Example 9 Rhizobia-Mediated Canola Transformation

A. Rhizobium Inoculum Preparation:

Rhizobia strains with pMON96033 were used for canola transformation. TheRhizobia strains with the vector from a glycerol stock were inoculatedinto 10 mls of TY medium with 50 mg/l kanamycin and 50 mg/lspectinomycin in a 50 ml Falcon tube and shaken at 28° C. overnight at200 rpm. The overnight Rhizobia culture was pelleted by centrifugationand resuspended in MG/L liquid medium. (MG/L broth: Mannitol 5 μl,L-glutamic acid 1 g/l, KH₂PO₄ 250 mg/l, NaCl 100 mg/l, MgSO₄.7H2O 100mg/l, biotin 1 μg/l, yeast extract 2.5 g/l, pH7.0). The OD₆₀₀ wasbetween 0.05-0.1.

B. Canola Explant Preparation and Co-Cultivation:

Canola transformation was done according to U.S. Pat. No. 5,750,871 andRadke et al., 1992. About 0.25 g of canola seed, cv. Ebony, wastransferred into a 1,5-ml Eppendorf tube and wetted with 95% ethanol. Tosterilize the seeds, 1 ml of 1% sodium hypochlorite solution was addedfor 30 min. The bleaching solution was replaced with distilled water andthe seeds were rinsed several times. The seeds were spread onto 1/10 MSgermination medium and kept in a Percival incubator at 24° C. with a 16hour light photo period.

Seed Germination Medium ( 1/10 MS medium): 1/10×MS minimal organicsmedium (Gibco BRL; final sucrose 0.3%), pyridoxine 50 μg/l, nicotinicacid 50 μg/l, glycine 200 μg/l, PHYTAGAR (Gibco Invitrogen) 6 g/l, pH5.8; 20). Etiolated seedlings from 7-14 days old cultures were used asthe explant source.

Explants were inoculated in 1×10⁸ bacteria/ml. Rhizobium suspension wasdrawn off, and the inoculated explants were placed onto co-cultivationplates on top of filter paper, and incubated for about 2 days at 24° C.in continuous light. Co-cultivated explants were assayed for gusexpression and found to contain blue spots indicating transformation ofcanola cells (FIG. 9).

Co-cultivation Medium (MS-1): MS salts (Caisson Laboratories, Logan,Utah), myo-inositol 100 mg/l, thiamine-HCl 1.3 mg/l, KH₂PO₄ 200 mg/l,2,4-D 1 mg/l, sucrose 3%, PHYTAGAR (Gibco Invitrogen) 7 g/l, pH 5.8.

C. Callus Induction:

Co-cultivated explants were transferred to Callus Induction (B5-1)medium. for 6 days at 24° C. in continuous light at ˜100 μE/m²/s. Fivestrains from M. loti, R. leguminosarum, S. fredii and S. meliloti showedefficient gene transfer into canola explants with frequency of guspositive explants ranging from 21% to 73% (FIG. 10).

Callus Induction Medium (B5-1): Gamborg's B5 salts (Caisson Labs), B5vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/lthiamine-HCl), 100 mg/l inositol, 1 mg/l 2,4-D, sucrose 3%,carbenicillin (PhytoTechnology, Shawnee Mission, Kans.) adjusted tofinal potency of 325 mg/l, 50 mg/l Timentin, 7 g/l PHYTAGAR (GibcoInvitrogen), pH5.8.

D. Shoot Regeneration and Selection:

Explants having callus were transferred to Shoot Regeneration medium(B5BZ) with AgNO₃ and incubated at 24° C. in continuous light of 100μE/mm²/s for 14 days. Explants were next transferred to ShootRegeneration medium (B5BZ) without AgNO₃. Shoots regenerated fromglyphosate-selected calli were harvested ˜every two weeks. An example ofearly shoots showing gus expression is shown in FIG. 11.

Shoot Regeneration Medium with Silver Nitrate (B5BZ+3Ag): Gamborg's B5salts (Caisson Labs), B5 vitamins (1 mg/l nicotinic acid, 1 mg/lpyridoxine-HCl, 10 mg/l thiamine-HCl), 100 mg/l inositol, BAP 3 mg/l(Sigma), zeatin 1 mg/l (Sigma), AgNO₃, 3 mg/l (Sigma), 45 mg/lglyphosate (Monsanto, 96.5% dry acid,), sucrose 1%, carbenicillin(PhytoTechnology) with potency adjusted to 325 mg/l, 50 mg/l Timentin,PHYTAGAR (Gibco Invitrogen) 7 mg/l, pH 5.8.

Shoot Regeneration Medium (B5BZ): Gamborg's B5 salts (Caisson Labs), B5vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/lthiamine-HCl), 100 mg/l inositol, BAP 3 mg/l (Sigma, zeatin 1 mg/l(Sigma), 45 mg/l glyphosate, sucrose 1%, carbenicillin (PhytoTechnology)with potency adjusted to 325 mg/l, Timentin 50 mg/l, PHYTAGAR (GibcoInvitrogen) 7 mg/l, pH 5.8.

E. Shoot Harvest:

Green shoots at least 0.5 cm in length were trimmed to isolate the mainaxis. Trimmed shoots were placed on Shoot Harvest medium (B5-0). Shootswere transferred to Rooting medium after 2 weeks.

Shoot Harvest Medium (B5-0): Gamborg's B5 salts (Caisson Labs), B5vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/lthiamine-HCl), 100 mg/l inositol, carbenicillin (PhytoTechnology) withpotency adjusted to 195 mg/l, sucrose 1%, PHYTAGAR (Gibco Invitrogen) 6g/l, pH 5.8.

F. Shoot Growth and Rooting:

Green shoots were transferred to Rooting medium (B5-0+2IBA). Shootsremained on Rooting medium until they formed roots Shoots weremaintained at 24° C., 16 hours light/day, ˜100 uE/m²/s.

Rooting Medium (B5-0+2IBA): Gamborg's B5 salts (Caisson Labs), B5vitamins (1 mg/l nicotinic acid, 1 mg/l pyridoxine-HCl, 10 mg/lthiamine-HCl), 100 mg/l inositol, IBA 2 mg/l (indole-3-butyric acid,Sigma) 150 mg/l cefotaxime (PhytoTechnology), sucrose 1%, PHYTAGAR(Gibco Invitrogen) 6 g/l, pH 5.8.

G. Transgene Detection and Transformation Frequency:

Total genomic DNA was extracted from greenhouse grown canola plants,digested with a single cutter BglII, and hybridized with DIG-labeled CP4probe. Four lines were confirmed to be transgenic (FIG. 11). Thetransformation frequency is summarized in the Table 6.

TABLE 6 Canola transformation frequency (TF) with Rhizobium strainsGUS/CP4 Strains Explants positive TF RL 2370G 150 2 1.33 SF 542 120 10.83 SM542C 120 1 0.83

Example 10 Rhizobia-Mediated Cotton Transformation Through Embryogenesis

A. Rhizobia Inoculum Preparation:

pMON101316 was electroporated into Rhizobia strains, verified byrestriction digestion of mini-prepared DNA and stored at −80° C. TheRhizobia strains with the vector from the glycerol stock were inoculatedinto 10 mls of TY medium with kanamycin (50 mg/l) and spectinomycin (50mg/l) in a 50 ml Falcon tube and shaken at 28° C. overnight at 200 rpm.The overnight Rhizobia culture was pelleted by centrifugation,resuspended in 20 mls of MS0 liquid medium and centrifuged again. Thepellet was resuspended in 20 mls of MS0 medium. The washed Rhizobia wasdiluted in MS0 to an OD₆₆₀ of about 1.0 for inoculation.

B. Explant Preparation:

Cotton transformation was done essentially according to U.S. Publ.2004087030. Seven days after seedlings were germinated, etiolated cottonseedlings from cultivar Coker were removed from a dark Percivalincubator. The hypocotyls from the PHYTATRAYs were harvested and placedin a sterile Petri dish containing sterile MSO to prevent the tissuefrom drying out. Hypocotyls were cut into small explants.

C. Inoculation and Co-Cultivation:

Using a sterile forceps, explants were transferred to sterile Petridishes, and Rhizobia inoculum was added. Explants were left in a sterilehood for 20 minutes, with swirling to ensure good contact of allexplants with the Rhizobia inoculum. The Rhizobia inoculum solution wasthen aspirated out and explants were gently blotted with sterile filterpaper. The inoculated hypocotyl pieces were placed onto culture plates.Co-culture of the plates of explants, covered with a plastic bag, wasperformed in a Percival incubator set at about 22-24° C., with a 10 hourlight/14 hour dark photoperiod for 2 days.

D. Stable Transformation Through Embryogenesis:

Two days post inoculation, cotton explant pieces were stained withX-gluc to test for GUS transient expression (FIG. 12). Blue spots inhypocotyls indicate the expression of the gus gene and transformation ofcotton cells. In order to obtain stable transformed plants, hypocotylexplants were transferred onto a plate containing UMSEL1629 selectionmedium, containing the appropriate selection agent. The plates were thencovered with PARAFILM and cultured 28° C. with a 16/8 hr. (day/night)photo period.

The stably transformed calli were confirmed by X-Gluc staining for gusexpression after 4 weeks on the selection medium (FIG. 13). Four weeksafter the initial transfer to selection medium, all the hypocotyls weretransferred to UMSEL 1788 medium, PARAFILMed and cultured for 7 days.Then the explants were transferred back onto UMSEL1629 for 4 weeks at28° C. with a 16/8 hr. (day/night) photo period.

Approximately 4 weeks after the second transfer on UMSEL1629, dependingupon the growth rate of the callus, clumps of calli were recovered andwere transferred to UMO plates. Individual plates are then labeled,covered with PARAFILM, and cultured at 28° C. in continuous dark.

Six to eight weeks after the callus has been on UMO the calli aresubcultured onto fresh UMO medium and cultured at 28° C. in continuousdark. After 6-10 weeks on UMO, embryogenic callus (EC) is ready to beharvested from independent callus lines on UMO and is transferred toTRP+ medium. Every 3-5 weeks, for approximately 3 months, activelygrowing tissue and small embryos on TRP+ plates are transferred to freshTRP+ medium and cultured at 28° C. in continuous dark. Embryos aretransferred to SHSU medium in Petri plates and covered with PARAFILM.Plates are cultured at 28° C. in a Percival with a 16/8 (day/night)photo period with maximum lighting (shelf and side lights). The embryosmay be subcultured on same medium one more time until germination.Plantlets recovered are cultured in a Percival or warm room at 28° C.with a 16/8 (day/night) photo period.

E. Molecular Analysis of Transgenic Nature of Cotton Calli Derived FromRhizobium-Mediated Transformation:

Genomic DNA was extracted from callus tissue, digested with a singlecutter BamHI, fractionated in 1% agarose gel, and transferred ontoHybond™ membrane (e.g. Appligen-Oncor, Illkirch, France; orAmersham-Pharmacia Biotech). A DIG labeled gus probe was used to detectthe presence of the transgene as an indicator of transformation. Sixlines of cotton calli, derived from S. meliloti, S. fredii and R.leguminosarum transformation with Ti helper plasmid, were found tocontain the gus gene (FIG. 14).

F. Media for Cotton Culture:

Recipe for 1 L of UMSEL—4.33 g MS salts, 2 ml 500× B5 vitamins, 0.1 ml2,4-D (1 mg/ml), 1 ml kinetin (0.5 mg/ml), 30 g glucose, pH 5.8, 2.5 gPHYTAGEL, 1.7 ml carbenicillin (250 mg/ml), 1 ml cefotaxime (100 mg/ml),plus selection agent: kanamycin 40 mg/L final concentration.Carbenicillin, cefotaxime and selective agents were addedpost-autoclaving.

Recipe for 1 L of UMSEL1788:—4.33 g MS salts, 2 ml 500× B5 vitamins, 0.1ml 2,4-D (1 mg/ml), 1 ml kinetin (0.5 mg/ml), 30 g glucose, pH 5.8, 2.5g PHYTAGEL, 1.7 ml (250 mg/ml) carbenicillin, 1 ml (100 mg/ml)cefotaxime, plus selection agent: kanamycin 40 mg/L final concentrationand 0.1 g sucrose dissolved in 100 ml water. Carbenicillin, cefotaximeand selective agents were added post-autoclaving.

Recipe for 1 L of UMSEL1629:—4.33 g MS salts, 2 ml 500× B5 vitamins, 0.1ml 2,4-D (1 mg/ml), 1 ml kinetin (0.5 mg/ml), 30 g glucose, pH 5.8, 2.5g PHYTAGEL, 1.7 ml (250 mg/ml) carbenicillin, 1 ml (100 mg/ml)cefotaxime, plus selection agent: kanamycin 40 mg/L final concentration.Carbenicillin, cefotaxime and selective agents were addedpost-autoclaving.

Recipe for 1 L of UMO—4.33 g MS salts, 2 ml 500× B5 vitamins, 30 gglucose, pH 5.8, 3.5 g GELRITE, 1.7 ml (250 mg/ml) carbenicillin, 1 ml(100 mg/ml) cefotaxime, 100 mg/l ascorbic acid, plus selection agent:kanamycin 50 mg/L final concentration.

Recipe for 1 L of TRP+—4.33 g/l MS salts, 2 ml 500× B5 vitamins, 1.9 g/lKNO₃, 30 g/l glucose, 0.1 g/l casein hydrolysate, 3.5 g GELRITE, pH 5.8.

Recipe for 1 L of SHSU—100 ml Stewart & Hsu Majors (10×), 10 ml Stewart& Hsu Minors (100×), 1.5 ml iron (100×), 10 ml Stewart & Hsu Organics(100×), 5 g glucose, 50 mg/l benlate, 2.2 g GELRITE, pH 6.8 (Stewart &Hsu, 1977).

Example 11 Rhizobia-Mediated Corn Transformation

A. Rhizobium inoculum Preparation and Media Composition:

pMON96036 containing CP4, GUS and gfp expression cassettes was used forcorn transformation. The vector was electroporated into various modifiedRhizobia strains, verified, and stored at −80° C. Rhizobia containingthe vector in a glycerol stock were streaked out on solid TY mediumsupplemented with antibiotics (kanamycin 40 mg/L and spectinomycin 31mg/L), and incubated at 28° C. for 2 days.

Two days before Rhizobia inoculation of the maize immature embryos, oneloopful of cells from a Rhizobia culture plate was inoculated into 25 mLof liquid TY medium supplemented with 62 mg/L of spectinomycin and 40mg/L of kanamycin in a 250 mL flask. The flask was placed on a shaker atapproximately 150-200 rpm and 27-28° C. overnight. The Rhizobia culturewas then diluted (1 to 5) in the same liquid medium and put back on theshaker. Several hours later, one day before inoculation, the Rhizobiacells were spun down at 3500 rpm for 15 min. The bacterial cell pelletwas re-suspended in AB-TY or ATA induction broth with 200 μM ofacetosyringone and 50 mg/L spectinomycin and 25 mg/L kanamycin and thecell density was adjusted to 0.2 at OD₆₆₀. The bacterial cell culture(50 mL in each 250 mL flask) was then put back on the shaker and grownovernight. On the morning of inoculation day, the bacterial cells werespun down and washed with liquid ½ MS VI medium (U.S. Publ. 20040244075)supplemented with 200 μM of acetosyringone. The bacterial culture wascentrifuged and the cell pellet was re-suspended in ½ MS PL medium (U.S.Publ. 20040244075) with 200 μM of acetosyringone and the cell densitywas adjusted to 1.0 at OD₆₆₀ for inoculation. Reagents are commerciallyavailable and can be purchased from a number of suppliers (see, forexample Sigma Chemical Co., St. Louis, Mo.).

B. Corn Embryo Isolation and Rhizobium Co-Cultivation:

For Rhizobia-mediated transformation, ears containing immature corn (Zeamays) embryos were isolated and transformed by bacterial co-culture asgenerally described by Cai et al. (U.S. Patent Application Publication20040244075), except that the immature embryos were isolated fromsurface sterilized ears and directly dropped into the prepared Rhizobiacell suspension. After the Rhizobia cell suspension was removed, theimmature embryos were transferred onto the co-culture medium (U.S. Publ.20040244075).

To investigate GFP transient expression, the co-cultivated corn embryoswere directly placed under a microscope with fluorescence light for GFPobservation. Alternatively, 10 randomly picked embryos afterco-cultivation were transferred into 1.5 ml Eppendorf tube and stainedwith X-gluc solution overnight at 37° C. for gus transient expression.FIG. 15 represents GUS transient expression of corn immature embryostransformed with five Rhizobium strains of R. leguminosarum, M. loti, S.fredii and S. meliloti compared to Agrobacterium tumefaciens strain ABIusing ATA induction medium.

It was noted that routine AB minimal medium used for Agrobacteriumgrowth and induction does not efficiently support Rhizobia growth.Rhizobia inoculums did not show significant growth in AB minimal mediumwithout any selection after one week shaking with 220 rpm at 28° C.Inclusion of 20 ml TY medium in AB minimal medium dramatically improvesRhizobia growth rate; while replacing glucose in AB-TY medium withL-arabinose and potassium gluconate in ATA medium decreasespolysaccharide production, resulting into tighter pellets. The change ofcarbon source in induction medium significant improves gus transientexpression with Rhizobia strains in corn.

C. Callus Induction and Regeneration of Transgenic Plants:

After co-cultivation, transformation was continued essentially asdescribed in U.S. Publ. 20040244075, with modifications of selectionconditions as appropriate. The embryos were transferred onto a modifiedMS medium (U.S. Publ. 20040244075) supplemented with 250 mg/Lcarbenicillin and 0.1 mM glyphosate Stably transformed calli with gypexpression were observed from the M. loti, S. fredii and S. melilotistains used in transient assay at this stage (FIG. 16). Representativetransformation frequencies are shown in Table 7.

TABLE 7 Corn transformation frequency with different Rhizobia strains.Induction Rhizobia Transgenic medium Strain Embryos plants TF AB ML4404177 3 1.69% induction ML542G 154 2 1.30% ABI 83 7 8.40% ATA ML542G 112 21.78% Induction SF4404 116 6 5.17% SF542C 124 2 1.61% SM542C 134 1 0.75%2370LBA 139 0 0% ABI 100 1 1%D. Molecular Analysis of Transgenic Plants Derived fromRhizobia-Mediated Transformation:

Total genomic DNA was isolated from greenhouse grown corn plants anddigested with a single cutter BamHI to estimate transgene copy number. ADIG-labeled gus probe was used to hybridize with the genomic DNA. Thetransgenic nature of putatively transformed tissues was confirmed forall lines but one (FIG. 17).

E. Germline Transmission of Transgenes in the Transgenic Corn Plants:

The flowering transgenic corn plants were either selfed or outcrossedwith the parental line of the corn genotype used for transformation(line LH244). Dry seeds were imbibed in water for 1 day for gus stainingor 2 days for gfp counting. gus or gfp expression and segregation in thetransgenic R1 seeds were confirmed (Table 8).

TABLE 8 Transgene expression in the transgenic corn R1 seeds. GFP +seeds/ Gus + seeds/ seed seeds Seeds Rhizobia Transgenic line # assayedassayed strain ZM_A8232 112 9/20 6/7 ML542G ZM_A8232/LH244 112 4/20ML542G ZM_A8234 48 0/10  0/20 ML542G ZM_A8246 133 12/20  5/5 ML542GZM_A8247 148 4/20 1/3 SF4404 LH244/ZM_A8247 150 13/20 SF4404 ZM_A8248 750/20 4/6 SF4404 LH244/ZM_A8249 104 13/20 SF4404 LH244/ZM_A8249 275  9/20SF4404 LH244/ZM_A8251 148  7/20 SF4404 LH244/ZM_A8251 123  5/20 SF4404LH244/ZM_A8251 108  4/20 SF4404 ZM_A8252 137 9/20 4/6 SF4404 ZM_A8255 7810/20  SM542C LH244/ZM_A8245 31  2/10 ML542G LH244/ZM_A8253 138 10/20SF542C LH244/ZM_A8253 67  5/20 SF542C LH244/ZM_A8254 72  8/20 SF542CLH244/ZM_A8254 100  6/20 SF542C LH244/ZM_A8235 81 12/20 ABILH244/ZM_A8235 116 13/20 ABI LH244/ZM_A8235 161  8/20 ABI ZM_A8237/LH244294 7/20 5/7 ABI ZM_A8244/LH244 250 4/20 ABI ZM_A8240 14 3/4 ABIZM_A8240/LH244 86 7/20 ABI ZM_A8238 31 10/10 ABI LH244/ZM_A8238 90 10/20ABI ZM_A8256 53 3/10 ABI LH244/ZM_A8256 55  8/20 ABI

Example 12 Rhizobia-Mediated Crop Transformation Through ConjugalTransfer System

An oriT-dependent plasmid conjugal transfer system in Rhizobium andrelated species may also be used to deliver a gene of interest (GOI)into plant cells and subsequently be integrated into the plant genome. Ahomogenous or heterogeneous conjugal transfer system could be used forthe gene transfer. Transgenic plants could then be regenerated withselectable markers through an established tissue culture system.Rhizobia strains may include Sinorhizobium spp., Mesorhizobium loti,Rhizobium leguminosarum and Rhizobium sp. NGR234, among others.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for transforming a corn plant cell, comprising: (a)contacting at least a first corn plant cell with a Rhizobiales bacteriumother than Agrobacterium sp, comprising: (i) a first nucleic acidcomprising a vir gene region of a Ti plasmid wherein the vir gene regionacts to introduce a nucleic acid of interest into the plant cell in aVirD2-dependent manner; and (ii) a second nucleic acid comprising one ormore T-DNA border sequence(s) operably linked to a nucleic acid ofinterest; and (b) selecting at least a first corn plant cell transformedwith the nucleic acid of interest.
 2. The method of claim 1, wherein thebacterium is a Rhizobia cell.
 3. The method of claim 2, wherein theRhizobia cell is grown in the presence of acetosyringone or othercompound that induces vir gene function prior to contacting the plantcell.
 4. The method of claim 2, wherein the Rhizobia cell is selectedfrom the group consisting of: Rhizobium spp., Sinorhizobium spp.,Mesorhizobium spp., Phyllobacterium spp., Ochrobactrum spp, andBradyrhizobium spp.
 5. The method of claim 4, wherein the Rhizobia cellis Rhizobium leguminosarum.
 6. The method of claim 5, wherein theRhizobia cell is R. leguminosarum bv, trifolii, R. leguminosarum bv,phaseoli or Rhizobium leguminosarum, bv, viciae.
 7. The method of claim1, wherein the plant cell is comprised in an explant from a plant seed,seedling, callus, cell suspension, cotyledon, meristem, leaf, root, orstem; and the explant is contacted with the bacterium.
 8. The method ofclaim 7, wherein the explant comprises an embryonic meristem; callus;cell suspension; cotyledon; or tissue from leaves, roots, or stems. 9.The method of claim 1, wherein the first and second nucleic acids areintroduced into the bacterium by electroporation.
 10. The method ofclaim 1, wherein selecting a plant cell transformed with the nucleicacid of interest is carried out in the absence of a selection agent. 11.The method of claim 1, wherein selecting a plant cell transformed withthe nucleic acid of interest comprises culturing the plant cell in thepresence of a selection agent, wherein the nucleic acid of interestconfers tolerance to the selection agent or is operably linked to afurther nucleic acid that confers tolerance to the selection agent. 12.The method of claim 11, wherein the selection agent is glyphosate,kanamycin, bialaphos or dicamba.
 13. The method of claim 12, wherein thenucleic acid of interest or further nucleic acid encodes EPSP synthase.14. The method of claim 13, wherein the EPSP synthase protein is CP4.15. The method of claim 11, wherein the selection agent is glyphosate.16. The method of claim 1, wherein the nucleic acid of interest is notphysically linked to a selectable marker gene.
 17. The method of claim16, wherein the marker gene and the nucleic acid of interest geneticallysegregate in progeny of a plant regenerated from the plant celltransformed with the nucleic acid of interest.
 18. The method of claim1, wherein the bacterium comprises at least a third nucleic acidcomprising a further nucleic acid of interest and wherein the plant cellis transformed with the third nucleic acid.
 19. The method of claim 1,further comprising regenerating a plant from the plant cell, wherein theplant comprises the nucleic acid of interest.
 20. The method of claim19, wherein regenerating a plant from the plant cell comprises inducingformation of one or more shoots from an explant comprising the plantcell and cultivating at least a first shoot into a whole fertile plant.21. The method of claim 19, wherein regeneration occurs byorganogenesis.
 22. A Rhizobia cell selected from the group consistingof: Rhizobium leguminosarum USDA2370, R. leguminosarum bv. trifoliiUSDA2048, leguminosarum bv. phaseoli USDA2668, Rhizobium etli USDA 9032,Sinorhizobium fredii USDA205, S. meliloti USDA 1002, and Mesorhizobiumloti USDA3471, the cell comprising (i) a first nucleic acid comprising avir gene region of a Ti plasmid wherein the vir gene region acts tointroduce a nucleic acid coding for a sequence of interest into a plantcell in a VirD2-dependent manner; and (ii) a second nucleic acidcomprising one or more T-DNA border sequence(s) operably linked to anucleic acid coding for a sequence of interest.
 23. The Rhizobia cell ofclaim 22, further defined as comprising a selectable marker.
 24. Themethod of claim 1, further comprising growing the bacterium other thanAgrobacterium sp, in a medium comprising carbon source(s) which minimizepolysaccharide production during growth in induction medium, wherein thecarbon source(s) used to minimize polysaccharide production duringgrowth in induction medium is glucose in AB-TY medium, or L-arabinoseand potassium gluconate in ATA medium.